The present study investigated the best photoperiod for culturing pacu (Piaractus mesopotamicus) in recirculation aquaculture systems (RAS) based on its growth performance and hematological and oxidative stress responses. Juveniles (∼ 5g) were subjected to five treatments (in triplicate): 24L (light):0D (dark), 15L: 09D, 12L:12D, 9L:15D, and 0L:24D for 45 days. A total of 225 pacu individuals were randomly distributed among 15 tanks of 210L (n = 15 per tank). Zootechnical, hematological (glucose, lactate, hematocrit, and hemoglobin), and antioxidant and oxidative stress parameters (glutathione S-transferase (GST), total antioxidant capacity against peroxyl radicals (ACAP), and lipid peroxidation (LPO) were analyzed. The zootechnical parameters (e.g., weight gain, Fulton's condition factor, and specific growth rate) were better and worse with 9L:15D and 24L:0D photoperiods, respectively. The hepatosomatic index was higher and lower in the 0L:24D and 9L:15D photoperiods. Blood lactate levels and antioxidant and oxidative stress responses were increased in the longest photoperiods (15L:9D and 24L:0D). In contrast, the treatments that showed lower oxidative damage (liver, gills, brain, and muscle) were 9L:15D and 12L:12D. In conclusion, manipulating artificial light is one way to improve fish growth and health, where the best photoperiod for pacu farming in RAS is 9L:15D.